veterinary sciences Article Cinnamon Aqueous Extract Attenuates Diclofenac Sodium and Oxytetracycline Mediated Hepato-Renal Toxicity and Modulates Oxidative Stress, Cell Apoptosis, and Inflammation in Male Albino Rats Gehad E. Elshopakey 1,* and Sara T. Elazab 2 1 Department of Clinical Pathology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt 2 Department of Pharmacology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt; [email protected] or [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +20-102-392-3945 Abstract: Among commonly consumed anti-inflammatory and antimicrobial drugs are diclofenac sodium (DFS) and oxytetracycline (OTC), especially in developing countries because they are highly effective and cheap. However, the concomitant administration of anti-inflammatory drugs with antibiotics may exaggerate massive toxic effects on many organs. Cinnamon (Cinnamomum zeylanicum, Cin) is considered one of the most broadly utilized plants with various antioxidant and anti- inflammatory actions. This study aimed to evaluate the possible protective effects of cinnamon aqueous extract (Cin) against DFS and OTC hepato-renal toxicity. Eight groups (8/group) of adult male albino rats were treated orally for 15 days with physiological saline (control), Cin aqueous extract (300 mg/kg b.w.), OTC (200 mg/kg b.w.), single dose of DFS at the 14th day (100 mg/kg b.w.), DFS + OTC, Cin + DFS, Cin + OTC, and Cin + DFS + OTC. The administration of DFS and/or Citation: Elshopakey, G.E.; Elazab, S.T. Cinnamon Aqueous OTC significantly increased (p < 0.05) the serum levels of alanine aminotransferase, aspartate amino- Extract Attenuates Diclofenac transferase, alkaline phosphatase, urea, creatinine, and uric acid. Serum levels of pro-inflammatory Sodium and Oxytetracycline cytokines, as well as hepatic and renal malondialdehyde and nitric oxide metabolites, were also Mediated Hepato-Renal Toxicity and raised following DFS and OTC administration. Meanwhile, the activities of reduced glutathione, Modulates Oxidative Stress, Cell superoxide dismutase, and catalase in liver and kidney were significantly suppressed in DFS, OTC, Apoptosis, and Inflammation in Male and DFS + OTC treated rats. Moreover, hepatic and renal tissue sections from these rats exhibited Albino Rats. Vet. Sci. 2021, 8, 9. overexpression of caspase-3 and cyclooxygenase-II on immunohistochemical investigation. The https://doi.org/10.3390/vetsci administration of Cin aqueous extract ameliorated the aforementioned deteriorations caused by DFS, 8010009 OTC, and their combination. Conclusively, Cin is a promising protective plant extract capable of attenuating the oxidative damage, apoptosis, and inflammation induced by DFS and OTC either Received: 25 November 2020 alone or combined, on hepatic and renal tissues. Accepted: 4 January 2021 Published: 6 January 2021 Keywords: cinnamon; diclofenic sodium; oxytetracycline; hepato-renal toxicity; oxidative stress; im- Publisher’s Note: MDPI stays neu- munohistochemistry tral with regard to jurisdictional clai- ms in published maps and institutio- nal affiliations. 1. Introduction Diclofenic sodium (DFS), a nonsteroidal anti-inflammatory (NSAID), analgesic and antipyretic drug, is widely used globally to relieve pain and fever [1,2]. It is extensively Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. consumed for the management of many chronic conditions such as rheumatoid arthritis, This article is an open access article osteoarthritis, ankylosing spondylitis [3]. DFS elicits its action by hindering the formation distributed under the terms and con- of prostaglandin through suppressing cyclooxygenase-1 (Cox-I) and cyclooxygenase-2 ditions of the Creative Commons At- (Cox-II) enzymes with the same potency [4].The presence of DFS as an over-the counter tribution (CC BY) license (https:// drug may lead to its abuse resulting in threatening deleterious effects on the liver, kidney, creativecommons.org/licenses/by/ and gastrointestinal tract [5]. The misuse of DFS has been contributed to gastrointestinal 4.0/). problems [6], hepatotoxicity [7–9], nephrotoxicity [10], and neurotoxicity [11]. Oxidative Vet. Sci. 2021, 8, 9. https://doi.org/10.3390/vetsci8010009 https://www.mdpi.com/journal/vetsci Vet. Sci. 2021, 8, 9 2 of 19 stress and mitochondrial damage are the main elements collaborating in DFS toxicity [12,13]. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly consumed in combination with antibacterial medications. However, the concomitant administration of NSAIDS with antibiotics may exaggerate the toxic effects of both drugs. Therefore, the effect of combining DFS and OTC on biological systems needs to be evaluated. Oxytetracycline (OTC), a member of tetracycline antibiotics, possesses high activity against a wide variety of micro-organisms, including Gram-positive and Gram-negative bacteria, in addition to chlamydia, rickettsia, mycoplasmas, and protozoan parasites [14]. It is commonly prescribed for the management of respiratory and skin diseases in human and livestock, particularly in developing countries for its antimicrobial efficacy and low cost [15]. The irrational utilization of OTC in excessive doses without medical guidance has harmful effects on the liver and kidney [15]. Prior report has shown that toxic doses of OTC caused vigorous microvesicular steatosis and even hepatic damage [16]. It has been recorded that OTC exhibits its hepato-renal toxicity through induction of membrane lipid peroxidation and reduction of tissue antioxidants biomarkers [17]. Several herbal medications are being evaluated for their mitigative effects and antioxi- dant activity [18,19]. Recently, great attention has been paid to the use of herbal remedies to treat many diseases [20]. Cinnamon (Cinnamomum zeylanicum, Cin) is considered one of the most broadly utilized plants in herbal treatment with various bioactive actions. It acts as a source of natural antioxidant for enhancing human health [21]. Cinnamon is characterized by its high content of polyphenolic compounds which serve as free radical scavengers [22]. Several pharmacological effects were recorded for cinnamon as anti-inflammatory, anti- microbial, anti-oxidant, and anti-diabetic effects [23–25]. It has been reported that total cinnamon extract may guard against cadmium, glutamate, bisphenol, gentamicin-induced oxidative injury [24–27]. This study was designed to assess whether DFS exaggerates the hepatorenal toxicity of OTC and whether cinnamon can guard hepatic and renal tissues against the hepatorenal toxicity caused by DFS and OTC. 2. Material and Methods 2.1. Plant Extract Preparation The cinnamon extract was prepared according to the method of Abdeen et al. [24] with some modifications. Dry cinnamon (Cinnamomum zeylanicum) bark was purchased and identified from Faculty of Agriculture, Mansoura University. Briefly, 10 g of cinnamon bark was cleaned, grounded with a mechanical grinder to form a fine powder. The powder was soaked in distilled water (100 mL). The mixture was boiled at 100 ◦C for 2 h. Then, it was filtered and dried overnight by heating in an oven at 80 ◦C. The resultant dry extract was weighed and kept for further analyses and administration. The yield percentage of crude cinnamon bark aqueous extract was about 20% (w/w). 2.2. Phytochemical Analysis Total phenolic content was performed according to a previous study by Wolfe et al. [28], using the Folin–Ciocateu’s reagent. Total flavonoid content was predestined following the protocol of Okada et al. [29] by using the aluminum chloride colorimetric method. The vanillin method was used to estimate total tannin content according to a previous report by Sadasivam and Manickam [30]. Additionally, the antioxidant activity was evaluated using the free radical scavenging method (DPPH) as reported by Thaipong et al. [31], then the maximum inhibitory concentration (IC50) was calculated using the exponential curve. 2.3. Chemicals Oxytetracycline was purchased from Cid Co., El Haram, Giza, Egypt. Diclofenic sodium was procured from Novartis Pharma company (El Amireya, Cairo, Egypt). Vet. Sci. 2021, 8, x FOR PEER REVIEW 3 of 20 2.3. Chemicals Oxytetracycline was purchased from Cid Co., El Haram, Giza, Egypt. Diclofenic so- Vet. Sci. 2021, 8, 9 3 of 19 dium was procured from Novartis Pharma company (El Amireya, Cairo Egypt). 2.4. Experimental Animals and Design Sixty-four2.4. Experimental adult male albino Animals rats and weighi Designng between 130–160 g were obtained from the laboratory animalSixty-four unit of Zagazig adult male University. albino ratsDuring weighing 14 days between of acclimatization, 130–160 g the were animals obtained from were fed normalthe laboratory control animaldiet and unit water of Zagazig ad libitum. University. The protocol During of 14this days experiment of acclimatization, was the accepted byanimals the Animal were Ethical fed normal Committee control of diet the and Faculty water of ad Veterinary libitum. The Medicine, protocol Mansoura of this experiment University,was Egypt accepted (approval by theno. AnimalR/2). The Ethical animals Committee were randomly of the divided Faculty into ofVeterinary the follow- Medicine, ing eight experimentalMansoura University, groups (nEgypt = 8); the (approval control group; no. R/2). received The animals physiological were randomly saline daily. divided into Cin groups;the treated following daily eight with experimental Cin aqueous groupsextract